Poxviruses provide a unique system for studying the replication of DNA. Required enzymes and factors are encoded within the viral genome and DNA synthesis and processing occurs within the cytoplasmic compartment of the cell. Therefore, it has been possible to apply genetic and biochemical approaches to the study of DNA replication. Our efforts has been towards ascertaining the structure and mode of replication of the poxvirus genome with particular emphasis placed on understanding the processing of the replicative intermediates including the telomere-like hairpin structure and the enzymes involved in its replication. The replication of vaccinia virus proceeds through concatemeric intermediates which are resolved into unit length DNA. Mutational analysis has demonstrated that a specific cis acting DNA sequence, highly conserved among poxviruses, as well as the palindromic structure of the concatemer junction, is essential for resolution, and that resolution occurred by conservative strand exchange. A model for resolution involving site-specific recombination and orientated branch migration is consistent with this data. We have identified proteins that are candidates for trans-acting components of telomere resolution and are characterizing their properties. A procedure has been developed for the construction of viral genomes containing large inserts of foreign DNA by ligation of subgenomic viral DNA fragments in the presence of the insert DNA. These DNA molecules are transfected into cells where they can be packaged into viral particles and subsequently propagated as virus.
